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Title: The evolution of immunoglobulin E
Author: Doré, Katy Alison
ISNI:       0000 0004 5989 8361
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2015
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Allergic asthma is a type I hypersensitivity reaction, mediated by IgE. Severe asthma is inadequately treated with existing medicines. A greater understanding of IgE structure and function, and how it relates to its receptors,FcεRI and CD23, is required to improve therapeutic intervention. An evolutionary approach has been used to further this understanding, with the investigation of three proteins that are evolutionarily related to human IgE: platypus IgE, chicken IgY and human IgM. The sequences of these proteins were compared and constructs were designed that contained different fragments of the constant regions of these antibodies. A (His)6-tag was attached to the C-terminus of IgM-Fc, Fcμ3-4 (fragment containingonly CH3-4domains), IgY-Fc, and platypus IgE-Fc, and these were successfully purified using two different methods: one using affinity columns specific to the protein and the other method using a nickel NTA column and size exclusion chromatography. The proteins were then checked using SDS PAGE analysis and Mass Spectrometry. The proteins were successfully crystallized and significant steps taken to improve the diffraction quality of the crystals. A crystal structure of human Fcε3-4 was determined at the highest resolution seen to date and a more extensive carbohydrate structure was observed. A detailed analysis of the flexibility of the Cε3 was performed using B-factors and a new method for comparing the quaternary structure of IgE is proposed and comparison made with all previously determined free and receptor bound structures. The thermal stability of the antibody structures was measured using Differential Scanning Fluorimetry to identify different unfolding characteristics for the Cε2 and Cε3 domains. Substantial progress towards the structure determination of IgM-Fc, which crystallised as a hexamer, is reported.
Supervisor: Sutton, Brian John ; Beavil, Andrew John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available